The purpose of this work is to understand the mechanisms involved in developing an effective immune reponse to a lethal infection with the fungi, Histoplasma capsulatum (H. Capsulatum). In addition, it is also our intent to develop experimental murine models of histoplasmosis in immunocompromised mice to study novel treatment regimens that might be effective in treating disseminated histoplasmosis infection in humans. Primary infection to H. Capsulatum often results in a self limited upper respiratory infection in humans; however, in immunocompromised hosts, disseminated infection can occur through reactivation of a previous infection. Since disseminated histoplasmosis has emerged as a difficult clinical entity in individuals infected with HIV, it was of interest to study the factors involved in maintaining an effective memory immune response. To this end, it was important to first establish a thorough understanding of the factors which regulate effective primary immunity. In this regard, previous work in this laboratory has shown in a murine model of disseminated histoplasmosis that IL-12, IFN or TNF were important factors in mediating primary protection. These studies were extended to study the role that these and additional factors such as nitric oxide and neutrophils had in maintaining an effective immune response following a secondary challenge with H. Capsulatum. A second series of studies were intiated in immunodeficient SCID mice to detemine if infected mice could be treated with combination cytokine therapy and chemotherapy.The work involved in this project entails infection of both normal and immunocompromised mice with varying doses of H. Capsulatum. At the time of infection, mice are segregated into various groups are treated with neutralizing antibodies against a range of cytokines ( i.e. IL-12, IFNg, or TNFa) or depleted of specific cell types ( i .e. T cells or neutrophils) to determine their importance in providing effective immunity. A protective response is assessed by mortality and a determination of the H.capsulatum burdern from liver or spleen cells as determined by quantitiative culture. In addition, PCR and cytokine specific ELISA are done from spleen cells to determine the mechanism by which a protective response is achieved and maintained.There were two major research findings uncovered over the last year. The first study showed that immunodefecient SCID mice infected with a lethal dose of Histoplasma capsulatum could be protected by combination treatment with IL-12 and Amphotericin B. It was further shown that this treatment could be given for only three weeks and still provide sterilizing immunity for as long as 90 days after treatment was discontinued. The mechanism by which this treatment induced protection was shown to be through an increase in both IFNg and TNFa. The second major finding was noted when our studies focused on the factors required to protect mice following secondary challenge with H. Capsulatum. Remarkably, the results showed that secondary immunity could be maintained in the absence of IFNg or TNFa. These data are of fundamental interest in showing that effective immunity to an intracellular infection can be achieved in the absence of IFNg through a TNFa dependent mechanism.As a follow up to these studies, since CD4 and CD8+ T cells have been shown to be important in immunity to H. capsulatum and they both produce IFN- g and TNFa, we were interested in determining the cellular source of these cytokines respectively. We first showed that mice depleted of CD4 or CD8+ T cells had accelerated mortality following primary infection. By contrast, in secondary infection depletion of either CD4 or CD8+ T cells did not affect the outcome. Thus, either cell type is important in primary infection. Since CD8+ T cells had a role in primary infection it was of interest to determine the mechanism by which these cells were mediated immunity. In this regard, perforin -/- mice were infected with a sublethal or lethal dose of H. capsulatum. Remarkably, mice injected with either dose had accelerated mortality. Furthermore, perforin -/- mice depleted of CD8+ T cells infected with a lethal dose of H. capsulatum had accelerated mortality and increased fungal burden. These data strongly suggest that perforin has a role in primary immunity to H. capsulatum. In addition, in additional experiments we shown that there is a perforin independent role, which is mediated through IFN-g or TNFa. Future research will be focused on demonstrating that TNFa can directly mediating intracellular killing of H. capsulatum.